The dentate gyrus and subventricular zone are permissive areas for neurogenesis in the mammalian brain, while most other areas are not. Much emphasis is being placed on finding the set of cues in these environments that differ between permissive and non-permissive settings. As these factors are elucidated it is becoming clear that many signaling molecules active in neurogenic zones are also present in non-permissive areas, particularly after injury. Why then do neural precursor cells respond by making neurons when exposed to some regions while not others? While there are a number of intracellular pathways that are known to modulate the efficiency and ability of neural precursors to produce neurons, there has been little consideration as to whether manipulation of any of these pathways are able to overcome nonpermissive environments in vitro or in vivo. In this proposal we will address the hypothesis that neural precursor cells can overcome non- permissive environments by manipulation of the basic pathways that control cellular fate specification. Specific Aim 1: Define the mechanism(s) underlying directed differentiation of neural precursor cells into neurons or oligodendrocytes in stem cell growth conditions in vitro. Forced expression of Neurogenins (Ngn) or Mash1 drive neuronal differentiation and Sox10 drives oligodendrocyte differentiation within 24 hours without any change in growth conditions. Our preliminary data supports the hypothesis that these manipulations drive multipotential NPC to become fate-restricted precursors. In this aim we will test the mechanistic basis of this process through several sets of experiments: 1) Does directed differentiation with transcription factors change the rate or extent of differentiation when infected cells are exposed to differentiating conditions? 2) Does expression of Ngn, Mash1 or Sox10 instruct differentiation toward the appropriate fate (neurons for Ngn and Mash1 and oligodendrocytes for Sox10) and restrict differentiation into alternative fates? 3) Are the effects of expression of these factors reversible? 4) Is there synergism between neurogenic factors and Pax6? Between Sox10 and Oligs? 5) What is the basis for morphologic differences seen in neurons produced by forced expression of Mash1 compared to Ngn? These experiments are designed to acquire a further understanding behind the mechanism of forced differentiation of neural precursor cells. Specific Aim 2: Determine if directed differentiation overcomes non-permissive conditions in vivo. We will engineer precursors to express Ngn1, Ngn2, Mash1 or Sox10 and transplant them into two conditions. First, we will inject them intraventricularly into the late embryonic mouse brain to test the behavior of these cells in a normally permissive environment. Second, we will inject the cells into the adult mouse striatum to test the behavior of these cells in less permissive conditions for neuronal or oligodendrocyte differentiation. Neural precursor cells present in the already formed brain present an exciting opportunity to promote regenerative repair of the nervous system either by harnessing the potential of endogenous precursors to expand and produce neurons or oligodendrocytes or by transplantation of dividing precursors that then respond to the injured environment to effect repair. If we are able to harness the regenerative capacity of these cells it will be possible to consider stem cell based treatments for multiple sclerosis, neurotrauma or neurodegenerative diseases. [unreadable] [unreadable]